The goal of this research project is to develop a PET radiotracer to quantify the vesicular acetylcholine transporter (VAChT). VAChT is a novel protein expressed in presynaptic cholinergic nerve terminals. This protein is a key biomarker for studying the loss of cholinergic neurons and synapses, which plays a major role in the cognitive dysfunction of dementia and is associated with a number of neurodegenerative diseases. A PET tracer for VAChT will provide a unique tool to measure the loss of cholinergic neurons and assess the severity of cognitive dysfunction of dementia associated with neurodegenerative diseases. In addition, it has been found that the level of VAChT in the brain is altered during treatment of drug abuse and psychosis. Therefore, a PET tracer for VAChT also will provide a new tool to monitor treatment of drug abuse and psychosis. Currently, PET measurement of brain VAChT in the brain has been hampered due to the lack of a suitable PET tracer. Therefore, it is extremely important to develop in vivo PET imaging agents for VAChT. To develop a PET radiotracer for imaging VAChT, we propose to explore a series of new novel carbonyl containing VAChT inhibitors and identify the more potent isomers of the lead compounds with nanomolar affinity (Ki < 10 nM) and high selectivity for VAChT by in vitro bioactivity determination. The identified isomers will be radiosynthesized with C-11 or F-18 and then will be validated in vivo rats and non-human primates. In addition, (-)-[18F]FBBV, a lead radio agent that belongs in this new class of VAChT inhibitors will be further evaluated to determine if it will be a suitable PET tracer for clinical imaging studies of VAChT. The initial in vivo validation of (-)-[18F]FBBV demonstrated high specific binding affinity in the VAChT enriched striatum of the brain. In the current project, we propose to further evaluate (-)[18F]FBBV and several structural congeners. There are five specific aims in this grant application: Specific Aim 1, Synthesis and Radiosynthesis. We will first resolve the racemic mixtures of 5 new lead compounds to determine which enantiomeric isomers are more potent and more selective using in vitro binding affinity screening assays; the more potent enantiomers will then be radiosynthesized with C-11 or F-18. Specific Aim 2, Biological evaluation in rats. Rat biodistribution and autoradiography studies will be conducted to determine if the radiotracers bind to VAChT-enriched striatum of the brain. Specific Aim 3, MicroPET imaging and blood metabolite analysis in nonhuman primates. Baseline studies, test-retest variability studies, specificity studies and reversibility/irreversibility studies of the 18F or 11C-labeled radiotracers will be conducted in macaques including metabolite analysis of arterial blood samples. Specific Aim 4, Dosimetry Studies. Whole body microPET imaging studies will be conducted in macaques to estimate the dose of radiation absorbed by different organs in human. Specific Aim 5, Toxicity Studies. Toxicity studies of the unlabeled form of the most promising PET radiotracer will be conducted. Specific Aims 4 and 5 are prerequisite steps for future PET imaging studies in humans.